Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase that regulates diverse pathways and cellular processes. Deregulation of PP2A is associated with many types of cancers and Alzheimer's Disease. PP2A is highly regulated at two major levels: trimeric holoenzyme controls substrate specificity; modulation of active site conformation regulates the level of enzyme activity. Our recent advance in understanding PP2A-specific methyltransferase LCMT-1 shows that PP2A methylation, a modification that enhances holoenzyme assembly, is stimulated by PP2A's phosphatase activity. Our studies further suggest compelling mechanisms for PP2A inhibitory protein a4 and PP2A phosphatase activator (PTPA), and point to hierarchy controls and a linear pathway of holoenzyme biogenesis: partially-folded PP2A is stabilized by a4 in an inactive form, and converted to an active form by PTPA; activated PP2A is then selectively methylated and enhanced to form substrate-specific holoenzymes. We recently made key breakthrough in crystallization of PP2A bound to a4 and PTPA, the highly dynamic complexes with highly regulated interactions. The research proposed here will combine x-ray crystallography with biochemistry, biophysics, yeast genetics, and cell biology to determine how a4, PTPA and PP2A methylation control PP2A structure and function to precisely drive holoenzyme biogenesis. We will determine the high-resolution structure of the PP2A-a4 complex, and address how their interaction controls PP2A stability and affects cell survival (Aim 1). We will determine the structural basis for the chaperone function of PTPA to the PP2A active site to gain insight into PP2A activation, controls of catalytic metal loading and substrate preferences, and elucidate mechanisms of PTPA in PP2A activation and cell survival (Aim 2). We will determine how defects in a4 and PTPA affect subsequent steps of holoenzyme biogenesis, and decipher the role of methylation in controlling holoenzyme structure, conformation and stability (Aim 3). These studies will reveal compelling mechanisms and hierarchy controls of holoenzyme biogenesis that restrict ambiguous phosphatase activity and ensure formation of active holoenzymes, which has a fundamental impact on cell cycle, survival, and drug sensitivity. PUBLIC HEALTH RELEVANCE: This proposal addresses critical gaps of our knowledge in a highly challenging and dynamic aspect of PP2A regulation, highlighting novel compelling mechanisms and hierarchy controls of holoenzyme biogenesis. This study will fundamentally advance our understanding of PP2A regulation, and provide long-awaited structural bases, novel principles and strategies of targeting PP2A that would benefit treatment of human diseases.